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Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans , we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes ( avr-14 and avr-15 ). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C . elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

Background The nematode Caenorhabditis briggsae has been used as a model in comparative genomics studies with Caenorhabditis elegans because of their striking morphological and behavioral similarities. However, the potential of C. briggsae for comparative studies is limited by the quality of its genome resources. The genome resources for the C. briggsae laboratory strain AF16 have not been developed to the same extent as C. elegans . The recent publication of a new chromosome-level reference genome for QX1410, a C. briggsae wild strain closely related to AF16, has provided the first step to bridge the gap between C. elegans and C. briggsae genome resources. Currently, the QX1410 gene models consist of software-derived gene predictions that contain numerous errors in their structure and coding sequences. In this study, a team of researchers manually inspected over 21,000 gene models and underlying transcriptomic data to repair software-derived errors. Results We designed a detailed workflow to train a team of nine students to manually curate gene models using RNA read alignments. We manually inspected the gene models, proposed corrections to the coding sequences of over 8,000 genes, and modeled thousands of putative isoforms and untranslated regions. We exploited the conservation of protein sequence length between C. briggsae and C. elegans to quantify the improvement in protein-coding gene model quality and showed that manual curation led to substantial improvements in the protein sequence length accuracy of QX1410 genes. Additionally, collinear alignment analysis between the QX1410 and AF16 genomes revealed over 1,800 genes affected by spurious duplications and inversions in the AF16 genome that are now resolved in the QX1410 genome. Conclusions Community-based, manual curation using transcriptome data is an effective approach to improve the quality of software-derived protein-coding genes. The detailed protocols provided in this work can be useful for future large-scale manual curation projects in other species. Our manual curation efforts have brought the QX1410 gene models to a comparable level of quality as the extensively curated AF16 gene models. The improved genome resources for C. briggsae provide reliable tools for the study of Caenorhabditis biology and other related nematodes.

Aim: Mediterranean terrestrial ecosystems serve as reference laboratories for the investigation of global change because of their transitional climate, the high spatiotemporal variability of their environmental conditions, a rich and unique biodiversity and a wide range of socio-economic conditions. As scientific development and environmental pressures increase, it is increasingly necessary to evaluate recent progress and to challenge research priorities in the face of global change. Location: Mediterranean terrestrial ecosystems. Methods: This article revisits the research priorities proposed in a 1998 assessment. Results: A new set of research priorities is proposed: (1) to establish the role of the landscape mosaic on fire-spread; (2) to further research the combined effect of different drivers on pest expansion; (3) to address the interaction between drivers of global change and recent forest management practices; (4) to obtain more realistic information on the impacts of global change and ecosystem services; (5) to assess forest mortality events associated with climatic extremes; (6) to focus global change research on identifying and managing vulnerable areas; (7) to use the functional traits concept to study resilience after disturbance; (8) to study the relationship between genotypic and phenotypic diversity as a source of forest resilience; (9) to understand the balance between storage and water resources; (10) to analyse the interplay between landscape-scale processes and biodiversity conservation; (11) to refine models by including interactions between drivers and socio-economic contexts; (12) to understand forest-atmosphere feedbacks; (13) to represent key mechanisms linking plant hydraulics with landscape hydrology. Main conclusions: (1) The interactive nature of different global change drivers remains poorly understood. (2) There is a critical need for the rapid development of regional-and global-scale models that are more tightly connected with largescale experiments, data networks and management practice. (3) More attention should be directed to drought-related forest decline and the current relevance of historical land use.

Abstract In Western Canada, approximately half of the calves produced are castrated before 1 wk of age. Therefore, it is important to identify effective analgesic drugs to mitigate pain associated with castration and consequently improve animal welfare. The aim of this study was to assess the efficacy of a single s.c. dose of meloxicam at mitigating pain associated with knife and band castration in 1-wk-old calves. Seventy-two Angus crossbred bull calves (47.3 ± 6.70 kg of body weight (BW), 1 wk old) were used in a 3 × 2 factorial design where main factors included castration method–sham (CT), band (BA) or knife (KN) castration– and medication–lactate ringer (NM) or 0.5 mg/kg BW of meloxicam (M). Measurements included different physiological and behavioral parameters. Samples were collected on day −1, immediately before castration (T0); and 60, 90, and 120 min and 1, 2, 3, and 7 d after castration except for visual analog scale (VAS) which was collected at the time of castration. The salivary cortisol concentrations were greater (P = 0.04) in KN and BA calves than CT calves 60 min after castration, while 90 min after castration BA had greater concentrations than CT calves. Substance P concentrations were greater (P = 0.04) in NM calves than M calves on d 3 and 7 after castration. The serum amyloid-A (SAA) concentrations were greater (P = 0.05) in KN calves than BA and CT calves on days 0, 2, and 3, while BA calves had greater SAA concentrations on day 7 than KN and CT calves. The visual analog scores were greater (P < 0.01) in KN calves than BA, and in BA compared to CT calves. The KN calves tail flicked more (P < 0.01) than BA and CT calves, and NM calves tail flicked more (P = 0.03) than M calves. No castration or medication effect (P > 0.10) was observed for stride length, walking, standing, lying ventral, eating, foot stamping, head turning, lying and standing percentage, performance, platelets, or body temperature. Overall, knife castrated calves exhibited a greater acute pain response than band castrated calves. Meloxicam was able to reduce substance P concentrations, white blood cell counts and number of tail flicks after castration, suggesting that the drug was able to mitigate acute pain to some extent. However, meloxicam did not have an effect on the other physiological and behavioral parameters assessed.

Three experiments were conducted to evaluate the effects of band and knife castration on behavioral and physiological indicators of chronic pain in beef calves at 3 different ages (36 calves/age group): 1 wk of age (Exp. 1, 4 ± 1.1 d of age, 43 ± 1.1 kg BW), 2 mo of age (Exp. 2, 63 ± 2.3 d of age, 92 ± 1.7 kg BW), and 4 mo of age (Exp. 3, 125 ± 4.6 d of age, 160 ± 3.4 kg BW). In each experiment calves were randomly assigned to either sham (CT), band (BA) or knife (KN) castration. Experiments 1, 2, and 3 ended when the testicles of banded calves had sloughed off (68, 49, and 42 d, respectively). Animal BW and rectal temperature were recorded weekly over the experimental period. Salivary cortisol, substance P, haptoglobin, scrotal area temperature using infrared thermography, visual evaluation of swelling (5-point scale), and gait stride length were collected on d -1 and immediately before castration and weekly thereafter until the end of the study. Hair samples were collected 1 d prior to and 28 d after castration and at the end of the study for cortisol concentration. Standing and lying behaviors were recorded over a 28-d period immediately after castration. No differences ( > 0.10) were observed in salivary cortisol, substance P, haptoglobin, or hair cortisol among castration methods for any of the 3 ages. No changes in behavior were observed in calves castrated at 1 wk or 2 mo of age. In 4 mo-old-calves, BA spent less time lying ( < 0.01) than CT and KN calves. Also, the average duration of lying time for BA calves was greater ( < 0.05) than for CT calves. Both, 1-wk- and 2-mo-old calves had inflammation in the scrotal area lasting 7 d after KN castration, whereas inflammation was observed for up to 14 d in 4-mo-old calves. Swelling in BA calves lasted for 21 to 28 d in the 2 younger groups of calves, whereas in 4-mo-old calves swelling was observed until d 35 postcastration. Knife- and band-castrated calves did not exhibit indicators of chronic pain or distress when the procedures were performed in calves younger than 2 mo of age. Therefore, pain mitigation should be used when castrating to improve animal welfare, especially when castrations are performed in calves older than 2 mo of age independent of the method of castration.

The newly revised Canadian Codes of Practice for the management of beef cattle requires that as of 2018, calves older than 6 mo of age be castrated using pain control. Castration is a husbandry procedure commonly done without pain control, and there is a lack of agreement on an effective pain mitigation strategy specific to castration. The aim of this study was to identify the optimal time of administration of meloxicam prior to castration. Thirty-four Angus and Angus crossbred bull calves (282 ± 28.0 kg BW) were randomly assigned to 1 of 3 treatments receiving a single s.c. injection of meloxicam (0.5 mg/kg BW): 6 h (6H; = 11), 3 h (3H; = 12), or immediately (0H; = 11) before knife castration. Measurements included visual analog scale (VAS), head movement (HM), accelerometer movement (AM) and strain gauge exertion force (EF) on the squeeze chute, stride length (SL), lying and standing behavior, salivary cortisol (SC), haptoglobin, serum amyloid A (SAA), substance P (SP), and scrotal temperature (ST). Samples were collected on d -7, -5, -2, -1, and immediately before castration (T0) and 30, 60, 120, and 240 min and 1, 2, 5, 7, 14, 21, and 28 d after castration, except for VAS, AM, EF, and HM, which were obtained at the time of castration. A time × treatment effect ( = 0.01) was observed for SP, where 0H had lower concentrations than 3H and 6H calves 1 d after castration, whereas 3H calves tended to have greater levels than 6H calves 5 d after castration. Mean ST was greater ( < 0.01) in 6H calves compared to 0H and 3H calves 120 min after castration, whereas 6H and 3H calves had greater ST compared to 0H calves 240 min after castration. On d 1 after castration, 6H calves had greater ST than 0H and 3H calves, whereas 0H calves had greater ST compared to 3H and 6H calves on d 28 after castration. The SL tended ( = 0.09) to be shorter in 3H and 6H calves than 0H calves 30, 60, 120, and 240 min after castration. Number of peaks from the AM between 2 and 3 SD above or below the mean were greater ( = 0.03) in 3H and 6H calves than in 0H calves. No treatment differences ( > 0.10) were observed for the number of peaks and area for AM and EF, VAS, HM, SC, or haptoglobin. On the basis of these results, the optimal time to administer s.c. meloxicam in 7- to 8-mo-old knife-castrated calves is immediately before castration (0H), as evidenced by fewer indicators of pain and inflammation compared to 3H and 6H calves.

Three experiments evaluated the effect of band and knife castration on acute pain for the first 7 d after the procedure on 1-wk-, 2-mo-, and 4-mo-old calves. All calves were blocked by age and weight and randomly assigned to 1 of 3 treatments: sham castration (control, CT), band castration (BA), and knife castration (KN). Experiment 1 evaluated 1-wk-old Angus bull calves ( = 34; 43.0 ± 6.61 kg BW), Exp. 2 evaluated 2-mo-old Angus bull calves ( = 34; 91.5 ± 11.93 kg BW), and Exp. 3 evaluated 4-mo-old Angus bull calves ( = 35; 157.6 ± 22.52 kg BW). For all experiments, physiological and behavioral parameters were collected before (d -1 and immediately before castration [T0]) and after (60 and 120 min and on d 7) castration to assess acute pain. Physiological measures included complete blood cell count, cortisol, substance P, and scrotal temperature. Behavioral measures consisted of a visual analog scale, stride length, and time and frequency budgets for walking, standing, lying, tail flicking, foot stamping, and head turning. Performance parameters included initial and final BW and ADG. In Exp. 1, tail flicking was greater ( = 0.02) in KN calves compared to BA calves 2 to 4 h after castration, although no differences were seen between BA and KN compared to CT calves. In Exp. 2, a treatment × time interaction ( = 0.02) was observed for cortisol, where KN calves had greater cortisol concentrations 120 min after castration compared to BA and CT calves; KN calves also lay down and ate less ( < 0.01; = 0.02) and stood and walked more ( < 0.01; = 0.05) compared to BA and CT calves 2 to 4 h after castration. In Exp. 3, a treatment × time interaction ( < 0.01) was observed for cortisol concentrations in which all treatments were different from one another at 60 and 120 min, with BA calves having the greatest concentrations, KN calves being intermediate, and CT having the lowest concentrations. Overall, KN and BA castrated calves presented physiological and behavioral changes associated with acute pain; however, 2-mo-old BA calves presented the fewest behavioral changes and no physiological changes associated with acute pain compared to CT calves.

Salvage logging is thought to have negative impacts on soil functionality because it may increase soil compaction and reduce vegetation cover and soil organic matter content. We investigated whether and to what extent burning and subsequent logging initially altered soil functionality of a Mediterranean forest of Pinus halepensis M. Soil functionality indicators (e.g. soil enzyme activities, basal soil respiration, glomalin-related soil protein, and microbial carbon) were measured in March and October 2017 in unburned forest plots, nearby plots severely burned by wildfire in July 2016, and nearby burned plots severely burned by wildfire and then logged in December 2016 using a lightweight agricultural tractor. The results showed significant differences among three groups: unburned soils sampled in spring (1) and autumn (2), and burned soils (not subject or subject to logging) sampled in spring and autumn. In unburned plots, seasonality had a significant effect, which disappeared in burned plots regardless of whether they had been logged. The burned plots had higher content of organic matter and total nitrogen than the unburned soils but they were not correlated to higher soil respiration or microbial biomass. There were not any differences in any of the soil functionality indicators between the unlogged and logged burned plots. In addition, the burned plots had a higher glomalin-related soil protein content than the unburned soil in the autumn measurement. Overall, the results suggest a short-term wildfire impact of soil properties whereas logging using a lightweight tractor produced no significant impacts in this sparse Mediterranean pine forest.

A method for the determination of total reducing capacity (TRC) based on the reduction of Cu(II) to Cu(I) by antioxidants in a buffered solution (pH 7.0) containing 4,4′-dicarboxy-2,2′-biquinoline acid (BCA) was developed. Absorbance values at 558 nm characteristic of the Cu(I)/BCA complexes formed were used to determine the TRC of aqueous extracts of twelve Brazilian plants. The TRC values obtained with the suggested method correlated well with values obtained using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (r2 = 0.959). They were also compared with the total polyphenol content (using the Folin-Ciocalteu reagent) and the good agreement (r2 = 0.919) indicates that the polyphenols should be responsible for this reducing capacity. The method proposed here (and successfully applied in plant extracts) can be used to measure the TRC of aqueous samples derived from other plants (e.g., teas, juices, beers and wines) and even in biological samples (e.g., serum, urine and follicular fluid). To achieve a structure-activity relationship of the proposed reaction, the reduction capability of 25 standard antioxidants (phenolic derivatives, flavonoids, stilbenoids, vitamins, etc.) was individually evaluated and the apparent molar absorptivity values (at 558 nm) obtained were compared and discussed.

Forty newly weaned beef calves (260 ± 32.6 kg) were transported 15 h in a livestock trailer (7.3 by 2.1 m) on 2 separate hauls 1 wk apart (20 calves/haul) to evaluate the effect of rest stop duration on indicators of calf welfare. Immediately following the 15-h journey, 15 calves/haul were randomly unloaded at a feedlot and randomly assigned to 1 of 3 rest stop treatments; calves without resting time (5 calves/haul) remained on the trailer and were used as the control group. Treatments included 0- (Control [CON]), 5- (RS5), 10- (RS10), or 15-h (RS15) rest periods in pens containing ad libitum access to water and long-stem hay. Following each rest period, calves were reloaded onto the same trailer and taken on another 5-h journey, before they were unloaded at the same feedlot, for a total transport event lasting 20 h. Control calves did not have access to feed or water until the end of the 20-h transit event. Behavioral measurements included bunk attendance (min/d) and standing and lying duration (min/d) recorded for 5 h after the 20-h transport event. Physiological measurements included saliva and hair cortisol, complete blood cell count, serum NEFA, haptoglobin, and substance P concentrations. All physiological measurements as well as BW were taken immediately prior to initial loading, at arrival at the feedlot after the 20-h event, and 48 h after the transport to the same feedlot. Hair cortisol was collected prior to the initial loading and 25 d after transportation. No differences ( = 0.78) in BW loss were observed among treatments after transportation. Standing time was greater ( < 0.001) in CON calves compared with RS5, RS10, and RS15 calves. Salivary cortisol was greater ( < 0.01) in CON and RS15 calves than in RS5 and RS10 calves at the end of the 20-h journey. Serum NEFA concentration was greater ( = 0.03) in RS5 and RS10 calves at arrival compared with CON and RS15 calves, but those differences were no longer observed ( = 0.49) 48 h after transportation. Concentration of substance P did not differ ( = 0.18) between treatments, and haptoglobin concentration tended to be greater ( = 0.07) in CON calves compared with the other treatments 48 h after arrival. Hair cortisol tended ( = 0.10) to be lower in RS5 calves compared with the other treatments. The results of this study indicate that rest stop periods ≥ 10 h did not prevent short- and long-term stress after transport in weaned calves.